Project Summary Capsular polysaccharides are essential to the virulence of many organisms, including Streptococcus pneumoniae, which is the major cause of community-acquired pneumonia and an important cause of sepsis, meningitis, and otitis media (middle ear infections). In this bacterium, capsules provide resistance against host- mediated opsonophagocytosis and are important in both infection and nasopharyngeal colonization. Ninety- eight distinct capsular serotypes have been identified, each consisting of unique combinations of sugars and linkages. All but two of the serotypes are synthesized by the Wzy polymerase-dependent mechanism in which repeat units built on the inner face of the cytoplasmic membrane are transported to the outer face of the membrane where they are polymerized into polysaccharide. Nearly all capsules in Gram-positive bacteria, and a large percentage capsules in Gram-negative bacteria, are synthesized by this mechanism. Knowledge of specific factors and mechanisms that could potentially disrupt capsule or its synthesis and thereby abrogate virulence is lacking. In studies of the Wzy pathway in S. pneumoniae, we detected a novel inhibitory activity that reduces capsule levels in multiple serotypes by more than ten-fold. Initial characterization showed the inhibitory component to be a small, heat stable molecule, likely a peptide. The inhibitory activity was present in a complex mixture that is readily available and amenable to biochemical, analytical, and physical analyses. The aims of the proposed project are to: 1) identify the inhibitor, and 2) initiate studies to determine the mechanism by which it affects capsule. The results of these studies will have particular significance to understanding novel mechanisms affecting capsule synthesis. Importantly, they could lay the groundwork for identifying inhibitors capable of reducing capsule levels during infection, thereby reducing virulence of the organism. Because mechanisms for synthesizing capsule are shared by many bacteria, the results may also have broad impact. Consequently, these and follow-up studies will have a high potential to positively impact point of care of patients infected by capsule-producing bacteria, including those resistant to antibiotics.